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Silva P.,University of Porto | Silva P.,The Interdisciplinary Center | Rocha M.J.,The Interdisciplinary Center | Rocha M.J.,Superior Institute of Health science North ISCS N | And 8 more authors.
Aquatic Toxicology | Year: 2012

In natural environments fish populations are exposed to many potential xenoestrogens, whereby understanding the impacts of mixtures continue to be of great interest. The main objective of this study was, therefore, to understand whether and how an environmentally relevant mixture of xenoestrogens found in the Douro River estuary can disrupt the normal gametogenesis in fish. For this purpose, adult zebrafish of both sexes were exposed for 21 days to an environmental mixture (MIX) of 11 xenoestrogens from diverse sources. A 100. ng/L ethinylestradiol (EE2) positive control was added. A quantitative (stereological) analysis with systematic sampling was made in the gonads, and using light microscopy both the relative and the absolute volumes of the gametogenic stages were estimated. Data point that the EE2 stimulus induced changes in structural compartments; with decreasing trends for the advanced maturation stages both in males and females. There was also a trend for a greater amount of interstitial tissue in males. Along with an interstitial fibrosis increase detected, the presence of a proteinaceous fluid was observed in both sexes and experimental groups (EE2 and MIX). Other histopathologic alterations were observed in the EE2 female group, such as the presence of foci of granulomatous inflammation and follicular mineralization in the germinal parenchyma and luminal areas. The most interesting finding of this study was that the exposure to the MIX caused a decrease of the relative volume of spermatozoa in zebrafish. This kind of estrogenic effect has not earlier been structurally quantified in such a fine detail with unbiased stereology in fish gonads. Despite the ultimate consequences of such disruptions being unknown, it could be logically argued that reduction or slowing-down of the appearance of the most mature cohorts and/or eventual interstitial fibrosis and other pathologic changes can adversely affect breeding. The findings add further explanatory bases for understanding the negative impacts of xenoestrogens. © 2012 Elsevier B.V.

Urbatzka R.,The Interdisciplinary Center | Rocha E.,The Interdisciplinary Center | Rocha E.,University of Porto | Reis B.,The Interdisciplinary Center | And 5 more authors.
Environmental Pollution | Year: 2012

In natural environments fish are exposed to endocrine disrupting compounds (EDCs) present at low concentrations and with different modes of actions. Here, adult zebrafish of both sexes were exposed for 21 days to an estrogenic mixture (Mix) of eleven EDCs previously quantified in Douro River estuary (Portugal) and to 100 ng/L 17α-ethinylestradiol (EE2) as positive control. Vitellogenin mRNA and HSI in males confirmed both exposure regimes as physiologically active. Potential candidates for estrogenic disturbance of steroidogenesis were identified (StAR, 17β-HSD1, cyp19a1), but Mix only affected cyp19a1 in females. Significant differences in the response of FSHβ, cypa19a2, 20β-HSD were observed between EE2 and Mix. Mtf-1 and tfap2c transcription factor binding sites were discovered in the putative promoter regions and corresponding transcription factors were found to be differentially expressed in response to Mix and EE2. The results suggest that "non-classical effects" of estrogenic EDC in fish are mediated via transcription factors. © 2012 Elsevier Ltd. All rights reserved.

Rocha M.J.,The Interdisciplinary Center | Rocha M.J.,Superior Institute of Health science North ISCS N | Rocha M.J.,Abel Salazar Biomedical Sciences Institute | Cruzeiro C.,The Interdisciplinary Center | And 3 more authors.
Toxicological and Environmental Chemistry | Year: 2012

Previous studies in the Douro River estuary, based on occasional sampling, showed the presence of several estrogenic disrupting chemicals (EDCs). In sequence, we hypothesized that such type of pollution was more likely an enduring issue than an occasional phenomenon, and that it may even affect recreational beaches in each side of the estuarine mouth. Thus to conclude about the continuous influx of EDCs, water samples were taken twice a day, once per a week, from March to May of 2009, at four sites within the estuary and at two sites in the coastline. After solid-phase extraction, the extracts were prepared for GC-MS analysis of 11 reference EDCs. These embraced natural and pharmaceutical estrogens (17β-estradiol, estrone and 17α-ethynylestradiol) and xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol, and their mono and diethoxylates and bisphenol A). Data showed the ubiquitous presence of potentially hazardous amounts of natural estrogens (particularly of estradiol, ca 5.5 ng L-1) and persistent organic pollutants such as nonylphenol mono (up to 550 ng L-1) and diethoxylate (up to 2000 ng L-1). It was concluded that the targeted area is continuously polluted by the assayed EDCs, and as a consequence, conditions exist for endocrine disturbance in the biota by chronic exposure to EDCs. © 2012 Copyright Taylor and Francis Group, LLC.

Rocha M.J.,The Interdisciplinary Center | Rocha M.J.,Superior Institute of Health science North ISCS N | Ribeiro C.,The Interdisciplinary Center | Ribeiro C.,Superior Institute of Health science North ISCS N | And 2 more authors.
International Journal of Environmental Analytical Chemistry | Year: 2011

This paper describes the development and validation of a GC-MS method which allows the simultaneous quantification of 11 endocrine disrupting compounds (EDCs) in surface water samples from both estuary and sea. The analysed EDCs are oestrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), 4-tertoctylphenol, 4-n-octylphenol, 4-nonylphenol, bisphenol A and finally, mono and diethoxylates of 4-nonylphenol and 4-octylphenol. The method includes the pre-concentration of water samples, 1000-fold factor, in OASIS HLB cartridges by solid phase extraction, the derivatisation of all EDCs by N,O-bis(trimethylsilyl) trifluoroacetamide added with 1% trimethylchlorosilane and pyridine (at 65°C for 30 min) and, finally the stabilisation of the EDCs-silylated derivatives, in hexane, for 72 h. The validation parameters revealed that this method was highly specific for all target compounds using real samples. The linearity of the calibration curves (r 2) showed correlation factors higher than 0.990. The detection limits ranged from 0.10 to 1.45 ng L -1, depending on each analysed compound, and recoveries were satisfactory for most of the assayed EDCs (460%). Analysis of samples from four polluted areas of Douro River estuary and from two points of the Atlantic Ocean (Portugal) showed high amounts of E1 (up to 1.96 ng L -1), E2 (up to 14.36 ng L -1) and EE2 (up to 2.76 ngL -1). © 2011 Taylor & Francis.

Carrola J.,University of Tras os Montes e Alto Douro | Santos N.,University of Porto | Rocha M.J.,University of Porto | Rocha M.J.,Superior Institute of Health science North ISCS N | And 4 more authors.
Environmental Science and Pollution Research | Year: 2014

Fish are bioindicators of water pollution, and an increased rate of their erythrocyte nuclear morphological abnormalities (ENMAs)-and particularly of erythrocyte micronuclei (EMN)-is used as a genotoxicity biomarker. Despite the potential value of ENMAs and MN, there is scarce information about fish captured in Iberian estuaries. This is the case of the Portuguese estuaries of the Mondego, Douro and Ave, suffering from different levels of environmental stress and where chemical surveys have been disclosing significant amounts of certain pollutants. So, the aim of this study was to evaluate genotoxicants impacts and infer about the exposure at those ecosystems, using the grey mullet (Mugil cephalus) as bioindicator and considering the type and frequency of nuclear abnormalities of erythrocytes as proxies of genotoxicity. Sampling of mullets was done throughout the year in the important Mondego, Douro and Ave River estuaries (centre and north-western Portugal). The fish (total n = 242) were caught in campaigns made in spring-summer and autumn-winter, using nets or fishing rods. The sampled mullets were comparable between locations in terms of the basic biometric parameters. Blood smears were stained with Diff-Quik to assess the frequencies of six types of ENMAs and MN (given per 1,000 erythrocytes). Some basic water physicochemical parameters were recorded to search for fluctuations matching the ENMAs. Overall, the most frequent nucleus abnormality was the polymorphic type, sequentially followed by the blebbed/lobed/notched, segmented, kidney shaped, vacuolated, MN and binucleated. The total average frequency of the ENMAs ranged from 73 ‰ in the Mondego to 108 ‰ in the Ave. The polymorphic type was typically ≥50 % of the total ENMAs, averaging about 51 ‰, when considering all three estuaries. The most serious lesion-the MN-in fish from Mondego and Douro had a similar frequency (≈0.38 ‰), which was significantly lower than that in the Ave (0.75 ‰). No significant seasonal differences existed as to the MN rates and seasonal differences existed almost only in the Douro, with the higher values in AW. In general, the pattern of ENMAs frequencies was unrelated with the water physicochemical parameters. Considering the data for both the total ENMAs and for each specific abnormality, and bearing in mind that values of MN in fish erythrocytes >0.3 ‰ usually reflect pollution by genotoxicants, it is suggested that mullets were likely being chronically exposed to such compounds, even in the allegedly less polluted ecosystem (Mondego). Moreover, data supported the following pollution exposure gradient: Mondego < Douro < Ave. The scenario and inferences nicely agree with the published data from chemical monitoring. © 2014 Springer-Verlag Berlin Heidelberg.

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